Ehrlichiae are obligate intracellular bacteria that predominantly infect bone marrow-derived cells in their mammalian hosts. Those species, for which a biological vector is known, are transmitted by ticks. Typically, Ehrlichiae are contained within a membrane-lined vacuole of their host cell.
Ehrlichiae species were initially characterized on the basis of host cell type, host species, and serologic cross-reactivity. Ehrlichiae may be divided into three phylogenetically distinct groups on the basis of nucleotide sequences of the 16S ribosomal RNA genes in each species and strong serologic cross-reactions. Each group is denoted by the historical precedent for the genetic group.
The Ehrlichia canis group includes 3 species known to infect predominantly monocytes and macrophages, and a single species known to infect canine granulocytes. Ehrlichia canis, the type species, E. chaffeensis, and E. muris infect mononuclear phagocytes of dogs, humans, and mice, respectively, while E. ewingii infects canine granulocytes (Ristic et al, In: Bergey's manual of Systematic Bacteriology, 1(9):1957 (1984)).
The second genetic group, the E. sennetsu group, includes E. sennetsu and E. risticii. They are monocytic Ehrlichiae that are agents of human Sennetsu fever of Japan and Potomac horse fever (equine monocytic ehrlichiosis) of horses worldwide.
The third group, the E. phagocytophila (Ep) genogroup, includes the granulocytic Ehrlichiae, E. equi, the agent of equine granulocytic ehrlichiosis (EGE) of horses, and an agent of canine granulocytic ehrlichiosis in the US, South America, and Europe; E. phagocytophila, the agent of tick-borne fever of ruminants in Europe; an as yet unnamed Ehrlichiae that is the causative agent of human granulocytic ehrlichiosis (HGE) in the United States and Europe; and more distantly, E. platys, a thrombocytic Ehrlichiae that causes mild cyclical thrombocytopenia in dogs.
Emerging genetic and antigenic data indicates that the members of the E. phagocytophila genogroup are very closely related or identical species (Chen et al, J. Clin. Microbiol., 32:589 (1994)). In humans, HGE was first recognized in 1990 (Chen et al, supra), and is considered an emerging disease of increasing clinical significance.
None of the granulocytic Ehrlichiae have been continuously propagated in vitro. This has continuously hampered the development of diagnostic tools for these infections, and investigation of the diseases and causative agents.
The clinical presentation of granulocytic ehrlichioses in man and animals are nonspecific and include fever, headache, rigors and malaise in humans, and fever, depression, and sometimes lameness in animals (Rikihisa, Clin. Microbiol. Rev., 4:286 (1991)). Ehrlichiae infections have dramatic effects on the hematologic and hepatic systems, and most infected humans and animal species develop leukopenia, thrombocytopenia, anemia, and evidence of mild hepatic injury. The appearance of membrane-bound vacuoles containing the pathogens within circulating leukocytes is suggestive of the diagnosis. However, human patients and some animals infrequently present with infected leukocytes in the peripheral blood.
Tetracycline antibiotics are the drug of choice for treatment of all ehrlichioses, and most human patients respond with a dramatic defervescence after therapy (Dumler et al, Clin. Infect. Dis., 20:1102 (1995)). In most cases, prevention of ehrlichioses focuses on vector control and prophylaxis using tetracyclines. The only agent for which a vaccine exists is E. risticii, which is cultivatable in vitro (Rikihisa, supra).
The exact economic toll extracted by the Ep group of Ehrlichiae is not known. In Great Britain, it is estimated that 2% of the entire goat population dies each year from secondary infections that occur only after E. phagocytophila infections (Brodie et al, Vet. Rec., 118:415 (1986)). In the United States, equine and canine infections with E. equi have been infrequently documented because of the lack of suitable diagnostic tools and nonspecific presentation of the illness. However, with the recent recognition that the agent of HGE is nearly identical genetically (Chen et al, supra), and antigenically (Dumler, J. Clin. Microbiol., 33:1098 (1995)), biologically with E. equi, and is capable of causing severe and fatal human infection, there has been an increased awareness of the prevalence of equine and canine infections. Serologic evidence of E. equi infection in some regions of California has been identified in up to 50% of the horses residing in those regions (Madigan et al, J. Am. Vet. Med. Assoc., 196:1962 (1990)). Similarly, nearly 20% of animals tested in a serosurvey of ill dogs in Oklahoma had evidence of E. equi infection (Rodgers et al, J. Vet. Diagn. Invest., 1:154 (1989)). Since the first identified case of HGE in 1990, there has been a logarithmic increase in the number of diagnoses of that human infection, especially since modern diagnostic methods have become available through specialized academic research facilities and some commercial laboratories. To date, approximately 115 cases of HGE have been recognized in the United States (Wormser et al, MMWR, 44:593 (1995)). Initial studies suggest that approximately 10% of human patients with Lyme disease may have been infected with the agent of HGE. Given the nearly 10,000 cases of Lyme disease reported annually in the United States, one would speculate that perhaps 1,000 of these patients may have also acquired undiagnosed HGE, in addition to those patients with HGE not accompanied by Lyme disease.
Ehrlichia phagocytophila and E. equi are transmissible through the bite of Ixodes ricinus (MacLeod et al, Parasitology, 25:273 (1933)) and I. pacificus ticks, respectively. Mounting evidence has implicated Ixodes scapularis (dammini) ticks as the vector responsible for transmitting the agent of HGE in the United States (Bakken et al, JAMA, 272:212 (1994)). It is assumed that larval ticks acquire the pathogen from a reservoir host, probably wild rodents, and that subsequent developmental stages, i.e., the nymphs and adults, transmit the Ehrlichiae during their blood meal.
Specific diagnosis of HGE, EGE, and tick-borne fever is accomplished by serology (Bakken et al, supra; Dumler, supra; and Madigan, supra), utilizing antigen prepared from infected animals. Diagnosis in animals is often suspected when typical intracytoplasmic inclusions are present in the peripheral blood leukocytes of febrile animals. In contrast, Ehrlichiae inclusions are variably present in the peripheral blood of humans who nevertheless may be very ill. As for other rickettsiae infections, the single most important determinant of clinical outcome is early diagnosis and early therapy with specific antimicrobial agents. If these patients are not treated promptly, the disease may quickly proceed to severe disease or a fatal outcome. Fatalities appear to be related to the development of secondary opportunistic infections that occur after the Ehrlichiae infection. Treatment with tetracycline antibiotics leads to a rapid therapeutic response (Bakken, supra).
Attempts to propagate E. equi and E. phagocytophila in vitro in primary neutrophils harvested from the peripheral blood of infected animals have yielded short term (48 to 72 hr) increases in the percentage of infected cells (Winjum et al, Vet. Microbiol., 34:355 (1993)). However, supplementation of the cultures with additional primary, uninfected neutrophils is unable to provide a continuous propagation system. This finding implies that mature neutrophils are probably not competent to become infected, but that new cells become infected as immature cells, probably in the bone marrow. A search for a susceptible mammalian cell line has been fruitless so far, and other cell lines known to support the growth of other Ehrlichiae species, such as DH-82, P388D.sub.1, U937, HEL, and Vero cells, do not support the growth of E. equi when co-cultivated with primary clinical samples.
Even more enigmatic is the biology of granulocytic Ehrlichiae in their tick vector. There is evidence, based on indirect immunofluorescent assays, that the organisms invade hemocytes of the tick, but other target tissues, if any, are unknown. By analogy with Anaplasma marginale, a related pathogen of cattle, it is expected that in the vector tick, a number of tissue types are invaded by E. equi and its relatives. Particularly, one can expect that the salivary glands of ticks should be invaded, as they would provide the rickettsiae with an obvious route of transmission to the mammal or to man. It is believed that culture systems for the production of the Ep group Ehrlichiae stages found in the mammal and in the vector would be valuable tools to elucidate the biology of these granulocytic Ehrlichiae in mammals, as well as in ticks, and would facilitate the development of appropriate diagnostic tools for HGE, and potentially an effective vaccine.
Because of the increasing prevalence and incidence of HGE and granulocytic ehrlichioses of horses and dogs, there is a need to develop reliable diagnostic tools that utilize antigen produced in vitro instead of in animals, such as horses. This capability will provide a more consistent, reproducible antigen for diagnosis, and will offer the ability for large scale production of both the mammalian stages and the vector stages in vitro.